REVIEW

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Next‐Generation Industrial Biotechnology‐Transforming

the Current Industrial Biotechnology into
Competitive Processes
Lin‐Ping Yu, Fu‐Qing Wu, and Guo‐Qiang Chen*

1. Introduction
The chemical industry has made a contribution to modern society by providing
The chemical industry has contributed al-
cost‐competitive products for our daily use. However, it now faces a serious
most everything to our daily lives due to the
challenge regarding environmental pollutions and greenhouse gas emission. With competitive processes for producing low‐cost
the rapid development of molecular biology, biochemistry, and synthetic biology, chemicals, materials, food additives, and
industrial biotechnology has evolved to become more efficient for production of fuels as well as pharmaceutics.[1,2] However,
chemicals and materials. However, in contrast to chemical industries, current it has been under heavy pressure because of
industrial biotechnology (CIB) is still not competitive for production of chemicals, the demand for sustainable development due
to excessive dependence on fossil resources,
materials, and biofuels due to their low efficiency and complicated sterilization emission of greenhouse gases (GHGs), and
processes as well as high‐energy consumption. It must be further developed into environmental pollution concerns.[3] The
“next‐generation industrial biotechnology” (NGIB), which is low‐cost mixed chemical industry is responsible for approxi-
substrates based on less freshwater consumption, energy‐saving, and long‐lasting mately 7% of global GHG emissions and
open continuous intelligent processing, overcoming the shortcomings of CIB and large amount of pernicious waste including
nondegradable plastics.[2] Therefore, the che-
transforming the CIB into competitive processes. Contamination‐resistant micro-
mical industry is required to develop green
organism as chassis is the key to a successful NGIB, which requires resistance to processes that match not only economic
microbial or phage contaminations, and available tools and methods for metabolic targets but also fulfill the societal demand for
or synthetic biology engineering. This review proposes a list of contamination‐ sustainability.[1,4]
resistant bacteria and takes Halomonas spp. as an example for the production of a Industrial biotechnology has therefore
variety of products, including polyhydroxyalkanoates under open‐ and continuous‐ attracted much attention for its sustainable
and environmentally friendly ways that may
processing conditions proposed for NGIB. enable the paradigm shift from petroleum‐
to bio‐based production.[5–7] However, cur-
rent industrial biotechnology (CIB) is not
economically competitive due to its high
Dr. L.‐P. Yu, Prof. F.‐Q. Wu, Prof. G.‐Q. Chen production cost associated with the complicated sterilization process
Ministry of Education Key Laboratory for Bioinformatics,
School of Life Sciences and low microbial transformation efficiency.[8–10] It becomes urgent
Tsinghua University to develop a low‐cost biotechnology, namely, the next‐generation
New Biology Building industrial biotechnology (NGIB), to overcome the disadvantages of
100084, Beijing, China CIB including easy contamination, consumption of large amount of
E-mail: chengq@mail.tsinghua.edu.cn
freshwater, complicated and expensive sterilization facilities and
Dr. L.‐P. Yu, Prof. F.‐Q. Wu, Prof. G.‐Q. Chen procedures, discontinuous processing, low product concentrations,
Center for Synthetic and Systems Biology
Tsinghua University and difficult product purification processes etc.[8,10] It is expected
New Biology Building that NGIB should be as similar as possible to the chemical
100084, Beijing, China industrial processes in terms of simplicity, yet maintaining its green
Dr. L.‐P. Yu, Prof. F.‐Q. Wu, Prof. G.‐Q. Chen processing features.
Tsinghua‐Peking Center for Life Sciences This paper reviews challenges, achievements, and prospects of
Tsinghua University NGIB to transform the CIB into more competitive processes,
New Biology Building
100084, Beijing, China highlighting potential microbial chassis, and successful examples of
NGIB for producing materials and chemicals.
Prof. G.‐Q. Chen
Manchester Institute of Biotechnology, Centre for Synthetic Biology
The University of Manchester
131 Princess Street 2. The Chemical Industry
Manchester M1 7DN, UK
Combined with a lower petroleum price, the chemicals,
DOI: 10.1002/biot.201800437 materials, or fuels produced by the chemical industries are

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cost‐competitive. The chemical industry is highly effective due

to the limited number of molecules that participate in the Lin‐Ping Yu is currently a Ph.D.
reactions under highly effective catalysts, high temperature, and candidate in Prof. Guo‐Qiang
high pressure.[11,12] This effectiveness includes high product Chen's laboratory at the Center for
concentration and purity, high substrate to product transforma- Synthetic and Systems Biology,
tion efficiency, and convenient product recovery methods.[11] School of Life Sciences, Tsinghua
However, chemical industry has several environmental bur- University. She obtained her B.S.
dens, including dependency on nonrenewable fossil resources, from the School of Life Sciences,
unsafe reaction conditions at high temperatures/pressure in the Xiamen University in 2014. Her
presence of explosive or flammable organic solvents, high‐ current research focuses on the
energy inputs during the production process, pollution caused development of functional PHA
by toxic industrial waste, and water as well as toxic or smelling biomaterials synthesized by engineered Halophiles.
gases, and nonrecyclable or nondegradable products[1,11,12]
(Figure 1). Therefore, it is highly desirable to maintain its Fu‐Qing Wu received his B.Sc. and
effectiveness while reducing its environmental impacts by Ph.D. from Southwest University in
developing green manufacturing processes. 2003 and China Agricultural
University in 2009, respectively.
3. Industrial Sustainability: Green Chemistry After receiving his Ph.D. degree, he
worked at the Chinese Academy of
“Industrial sustainability” can be understood as achieving Agricultural Sciences in Beijing and
sustainable production and processing within the context of joined the School of Life Sciences of
ecological and social sustainability.[1] Due to the mentioned Tsinghua University in 2018. His
disadvantages of the chemical industry, the concept of “green work focuses on studying the salt‐
chemistry” has been introduced to “efficiently utilizes (pre- resistant mechanism of halophilic bacteria and the medical
ferably renewable) resources, eliminates wastes, and avoids the application of PHA materials.
use of toxic and/or hazardous reagents and solvents in the
manufacture and application of chemical products.”[13] Green Guo‐Qiang Chen received his B.Sc.
chemistry has made large leaps forward to improve the and Ph.D. from South China
sustainability of industrial processes.[4,14] University of Technology in 1985
Simultaneously, there has been an explosion in the use of and Graz University of Technology
biotechnology in the last 30 years due to its excellent ability to (Austria) in 1989, respectively. He
control the power of life of microorganisms or enzymes to also conducted research in
realize a safe, effective, and environmentally friendly produc- 1990–1994 as a postdoc at the
tion process, which will continuously achieve immeasurable University of Nottingham in the UK
impact in this area.[1,15,16] The main purpose of industrial and the University of Alberta in
biotechnology is to design and develop bioprocesses for Canada. After joining Tsinghua
production of chemicals, materials, and biofuels to partially University in 1994, Professor Chen has been actively
replace petroleum‐based products.[12,15] It is primarily based on promoting the microbial Bio‐ and Material Industries in
the natural or engineered abilities of cell factories and enzymes China. Beginning from 2015, he became the Funding
to transform a wide range of renewable bio‐based raw Director of the Center for Synthetic and Systems Biology,
substrates into a variety of products[15,17] (Figure 1). Tsinghua University. His research is focused on
However, CIB is still unable to compete with chemical bioproduction and applications of PHA.
industries in terms of frequent disadvantages including low
productivity, low product concentration, unwanted by‐products,
not optimal metabolic pathways, high recovery costs, and (oil, fat, glycerol, and celluloses), and wood (lignocellulose and
difficult control of bioprocessing.[12] cellulose) for chemical production.[12,21] These organic agricul-
ture residues are available worldwide in large amounts and can
4. Current Industrial Biotechnology be conveniently transformed into important biofuels, chemical,
biomaterials and pharmaceutics.[12,22]
4.1. Utilization of Renewable Substrates

Efforts to conduct research on carbohydrates of agriculture

products or gases such as CO2 as sustainable carbon sources
(C‐sources) have been made.[18–20] In the United States, the
substitution percentage of fossil fuel with renewable resources
is envisioned to be 90% by 2090.[15] Industrial biotechnology
employs microorganisms grown on treated agricultural sub-
strates such as sugar beets and sugar cane (sucrose), starch
plants (starch, inulin, carbohydrates, and celluloses), oil plants
4.2. Metabolic Engineering and Synthetic Biology for
Strain Improvements
Metabolic engineering and synthetic biology approaches have
been used to improve productivity for diverse products since
1990s.[23] Most of the studies and applications have been
focusing on engineering the traditional chassis mentioned

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Figure 1. Comparison of A) chemical industry and B) industrial biotechnology. Chemical industry relies on fossil fuels, strict reaction condition, high‐
energy input, and will cause severe pollution by toxic industrial waste and water; while industrial biotechnology uses renewable bio‐based materials,
which can be conducted under mild conditions and the product can be easily treated.
above.[24–26] The engineering efforts have led to increasing
product titers, higher productivities, diverse products from one
chassis, widened substrate ranges, easier downstream proces-
sing, controllable product formations, higher ratios of substrate
to product formation, and more tolerance to stress.[27,28] The
reconstitution of genetic clusters in heterologous hosts has
often been used to improve the production performance or
diversify the product spectrum over the native producers.[29]
Recently, Escherichia coli (E. coli) has successfully overexpressed
nearly 30 genes to produce vitamin B12 via an engineered de
novo aerobic biosynthetic pathway.[30] To optimize the flux
through metabolic pathways, the modulation of enzyme
expression using a synthetic protein scaffold to improve the
pathway flux has been demonstrated.[31] In addition, long‐
durable stability of hosts expressing complicated pathways has
also been successfully developed.[32]
4.3. Successful CIB for Chemical Productions
CIB meets the demand of sustainability as it is based on
renewable resources, offering exceptional selectivity without the
need of protection and deprotection steps commonly required in
chemical processes.[33] It reduces energy consumption, waste
generation, and risks of explosion or fire accidents.[12] As a
possible replacement of chemical reactions, a variety of successful
CIB has been established for large‐scale productions.[34]
Many industrial microorganisms have been engineered as
excellent production strains for CIB, such as Bacillus subtilis,[35]
E. coli,[36] Corynebacterium glutamicum,[37] Pseudomonas puti-
da,[38] Saccharomyces cerevisiae (S. cerevisiae) and so forth.[39]
Food supplements and fine chemicals such as vitamins,[40]
steroids,[41] amino acids;[42] biofuels including ethanol,[43]
butanol, biodiesel,[44] and hydrogen;[45] materials such as
polyhydroxyalkanoates (PHA)[46,47] and polysaccharides;[48,49]
polymer precursors like lactic acid (LA)[50] succinic acid,[51]
and so forth. have been produced via those microorganisms.
Some CIBs are carried out exclusively using enzymes including
wild‐type, engineered, or evolved hydrolases such as acylases,
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esterases, lipases, cellulases, or proteases.[52] CIB based on
enzymes has been successfully employed to produce high‐value
products in pharmaceutical industries like blockbusting ator-
vastatin and many drug intermediates.[53]
4.4. Green Downstream Processing
CIB downstream processing includes separation of cell mass
and broth, product isolation and purification. A variety of in situ
product recovery techniques and applications have been
developed by integrating the downstream processing with CIB
bioprocessing.[54,55] Centrifugation and membrane technology
are the primary recovery steps used to separate biomass and
liquid broth.[56] Other primary isolation and purification steps
include solvent extraction, crystallization, precipitation, ultra-
filtration, or/and electrodialysis and the secondary separation
processes that involve fine microfiltration, distillation, recrys-
tallization, and chromatography, respectively, or in various
combinations.[17] As CIB mostly uses water as a medium
solvent, downstream processing usually generates nontoxic
wastewater. In real cases, organic solvents are used to extract
CIB products such as some antibiotics from broth or from the
cells, yet the organic solvents can be recovered by distillation
and no waste solvent needs to be disposed.[55,57]
5. The CIB Challenges
Although the CIB has overcome some disadvantages of the
chemical industry, it still faces several challenges, especially the
economic and technological aspects as discussed below.[9,10]
First, the price of basic raw substrates such as glucose from
hydrolysis has increased constantly, leading to higher produc-
tion costs.[58] Second, a large amount of freshwater is consumed
for preparing the growth medium, culturing cells, washing
production facility, extracting, and purifying final products. The
wastewater cannot be recycled resulting in additional waste-
water treatment costs.[10] Third, most CIB processes are
performed discontinuously over a short period of time in a
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batch or fed‐batch process to avoid contamination risk, which
results in low production efficiency.[59] The discontinuous
processes are complicated and must employ well‐trained
engineers to precisely control the whole system and restart
each cycle of batch/fed‐batch processes, further increasing the
process complexity and thus the labor cost.[60] Fourth, steriliza-
tion for preventing any possible contamination accounts for a
large part of production cost in CIB.[61] Last, the downstream
processes need continuous centrifugation, filtration, microfil-
tration, and/or membrane separation to purify products. These
processes are not stable as they are prone to plug, limiting the
effectiveness of CIB.
Therefore, it is very urgent to develop a green, efficient, and
economical technology with reduced substrate, water, and
energy consumption as well as high yields to make industrial
biotechnology much more competitive.
on less freshwater consumption, energy‐saving, and long‐
lasting open and continuous intelligent processing[8] (Table 1).
Instead of using expensive pure substrates as C‐sources, NGIB
should be based on low‐cost mixed substrates, which can
significantly reduce the production cost. Seawater, as a widely
available and inexhaustible water source, could be a sustainable
alternative of freshwater in NGIB. Furthermore, it is of great
importance to develop open and unsterile fermentation processes
that do not consume high energy for the sterilization procedure.
NGIB also needs to be conducted under a long‐lasting continuous
condition with higher production efficiency. Meanwhile, NGIB
should be composed of intelligent bioprocesses that could be
conveniently controlled by artificial intelligence (AI) instead of
complicated handling and monitoring processes.

6.2. NGIB Based on Low‐Cost Mixed Substrate and Seawater

6. Next‐Generation Industrial Biotechnology
6.1. Definition of NGIB
The concept of “NGIB” is defined to overcome the disadvan-
tages of CIB. NGIB should be low‐cost mixed substrates based
The commonly used C‐sources in CIB such as sugars or fatty
acids are expensive, leading to high production cost.[58,75] By
using low‐cost mixed substrates such as kitchen wastes, the
production cost can be significantly reduced.[76] For example,
PHA and hydrogen have been successfully produced by waste

Table 1. Current situations, future goals, and strategies required for NGIB.

Items Current situations Future goals Strategies References

Substrates Single C‐source
Water Freshwater
Process wastewater To wastewater treatment
plants
Processing Batch process
Facility Mostly expensive equipment
and control devices made
of stainless steels
Sterilization Hot and high‐pressure steam
Fermentation conditions Narrow growth conditions
Phage contaminations Frequent contamination
Energy consumption for oxygen Heavy consumption
supply
Separation of cells and broth Continuous centrifugation or
filtration
Substrate conversion ratio Low
Growth Slow
Control Difficult and complete
automation
Mixed C‐sources such as kitchen
wastes or shale gases, or CO2 or
hydrolyzed activated sludge
Seawater or household wastewater
Recycling wastewater
Continuous process
Low‐cost plastic, ceramic, or
cement bioreactors
Unsterile
Flexible growth conditions and AI
control
Resistant to phage contamination
Improvement on microbial O2 uptake
Sedimentation
High
Fast
AI control based on big data
[65,66]
Constructions or screening of multiple
substrate or mixed gases utilizing
microorganisms
[64–66]
Screening and construction of halophiles
or robust microorganisms
[65,67]
Construction of self‐flocculating
microorganisms
[59,64,66]
Contamination‐resistant
microorganisms
[68]
Employ contamination‐resistant
microorganisms
[66,68,69]
Contamination‐resistant
microorganisms in open processes
[65,70]
Contamination‐resistant and robust
microorganisms grown under a wide
range of temperature, pH, and
substrates
[59]
Phage resistant microorganisms
[65,71]
Periplasmic expression of hemoglobin
gene for better O2 uptake
[72]
Morphology engineering for large cell
sizes benefiting sedimentations
[65]
Removal or weakening competing
pathways
[73]
Changing growth patterns
[8,74]
Collection and analysis of historical data

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substrates under unsterile conditions.[77] The heat lysed
activated sludge can also be used as carbon and energy sources
under specific conditions, allowing cost reduction.[78] These
examples provide an economical way of using low‐cost mixed
substrate for developing NGIB.
Regarding a large amount of freshwater consumption, it has
been reported that there is no significant difference between the
growth of S. cerevisiae on freshwater or in seawater during
growth on pretreated date palm leaflets.[79] Studies have shown
that Halomonas campaniensis (H. campaniensis) is able to grow
in artificial seawater for poly(3‐hydroxybutyrate) (PHB) produc-
tion[64] and the seawater can also be used for xylose dehydration
to form furfural.[80] These attempts have proven that seawater
could be a promising alternative for NGIB.
6.3. NGIB Based on Open Continuous and Intelligent Processes
Microbial contamination is one important issue to CIB microbial
fermentation processes. The most common sterilization method
is heating due to its reliability and ease of use, while the high‐
energy consumption during heat sterilization significantly in-
creases the production cost.[61] The facilities both for medium and
air supply are very complicated, which result in high fixed asset
investment and energy consumption for CIB, reducing its
competitiveness to some extent. Open fermentation can be a
potential strategy lowering the related production cost in NGIB
(Figure 2). For example, thermophilic bacteria with the ability of
insensitivity to contamination under higher temperature, e.g.,
Bacillus coagulans, has been used to produce LA with a titer of
118 g L−1 and yield of 97.3% under high temperature;[81,82]
halophilic bacteria, like Halanaerobium saccharolyticum, with
excellent tolerance to high salt concentrations has been cultured
for the production of hydrogen and 1,3‐propanediol from crude
Figure 2. Comparison of A) CIB and B) NGIB. CIB employs a very strict procedure to prevent microbial contaminations including complicated
sterilization of the culture medium and air; while NGIB does not require the complicated sterilization processes; simple operations and AI control can
guarantee production.
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glycerol.[83,84] Although these studies showed the applicable
approaches to conduct open fermentation in NGIB, the possibility
such as biosafety containment should also be considered to
prevent potential regulatory problems, while for bulk chemical
production this is an issue of less concern.
Batch fermentation usually requires repeated fermentation
preparation and inoculation, which reduces the risks of
microbial contamination while the process efficiency is
low.[85] Compared with the batch fermentation, continuous
process is conducted by adding the substrate and harvesting the
final product continuously and simultaneously, leading to
economic advantages including the following: i) not requiring
sterilization of facilities in each round, and the residual
substrates can be recycled, cutting the energy and substrate
costs; ii) prolonging the microbial exponential growth, reducing
the processing time; and iii) eliminating the inhibition of toxic
by‐products in a timely manner, ensuring high yields of the
final product.[59,60,86] The continuous fermentation processes
have been applied successfully for production of ethanol,[87]
PHA,[64] hydrogen,[88] LA,[89] and protein.[90] It will be further
developed for more products in NGIB.
For the separation and downstream treatment of products,
the self‐flocculating strategies have been applied successfully.
For instance, S. cerevisiae and Zymomonas mobilis have been
used to improve productivities for ethanol production.[91,92]
Recently, a wastewaterless fermentation strategy has been
developed based on a self‐flocculating H. campaniensis strain.[67]
Morphology engineering for large cell sizes benefiting sedi-
mentations were also developed for convenient separation
process in NGIB.[72]
Furthermore, AI control based on robust microorganisms,
which can grow under a wide range of temperatures, pH, and
substrates, will be realized in NGIB, enabling an intelligent
bioprocess with reduced labor cost and enhanced production
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efficiency (Figure 2).[8] Several practical techniques have been
tested to optimize the medium composition for the production
of cholesterol oxidase (COD) by a Streptomyces sp.[74]
In most cases, the feasibility of open fermentation depends
on the unique properties of the production strains.[61] The
continuous fermentation meets a big challenge, which is the
higher risk of contamination, requiring the application of
contamination‐resistant strain.[59] At the same time, the
processing of wastewater, convenient separation of products,
and AI control also demand robust microorganisms to solve
various problems. Therefore, exploiting a robust, contamina-
tion‐resistant strain will be the key to meet the basic industrial
requirements for developing NGIB.
6.4. NGIB Based on Contamination‐Resistant Microorganisms
Possible contamination‐resistant microorganisms for the NGIB
could be selected from the following circumstances: 1) rapid
growth at very low or high pH, temperatures, osmotic pressure;
2) rapid growth on some toxic compounds (methanol, ethanol,
butanol, and heavy metals); 3) rapid growth on some unusual
substrates (cellulose, chitin, and rubbers) or even gaseous
substrate (H2, CO, CO2, and CH4);[8] and 4) convenient gene
manipulation methods and complete genome information
should also be available for engineering these organisms.[6]
Fortunately, some extremophilic microorganisms thriving in
the harsh environments meet some of the above requirements
and seem to be ideal platforms for developing NGIB.
Extremophilic microorganisms are widely distributed and are
a functionally diverse group that includes acidophiles, alkalo-
philes, psychrophiles, thermophiles, methanotrophs, metalo-
philes, xerophiles, and halophiles, among others (Table 2).[105]
Acidophiles can thrive in acidic or sulfuric environments.[93]
They produce extracellular enzymes like proteases, amylases,
cellulases, xylanases, and esterase with activities at low pH.[106]
The ability to grow at low pH prevents other microbial
contamination as few microorganisms could also grow under
such acidic conditions.[107] Meanwhile, alkalophiles grown at
high pH environments are potential contamination‐resistant
microorganisms, such as Bacillus marmarensis, has been
applied to produce ethanol at titers of 38 g L−1 and 65% yields
in unsterile media.[65]
Psychrophile bacteria produce extracellular hydrolytic en-
zymes such as lipase, chitinase, and pectinase that have high
catalytic efficiencies at lower temperatures and are useful in
many biotechnological fields.[94] Thermophiles that are able to
grow at high temperatures provide potential for open and
contamination‐free bioprocessing, such as Bacillus spp. and has
been applied to produce 210 g L−1 of L‐LA at around 50 °C under
open fermentation processes.[108] Thermoanaerobacter BG1L1
with high ethanol tolerance has also been employed to produce
ethanol under 70 °C.[95] It appears to be increasingly attractive to
develop NGIB based on thermophilic strains.
Xerophilic bacteria including Amycolatopsis, Saccharothrix,
and Streptomyces are reported to grow in arid habitats.[96]
Molecules produced by xerophilic bacteria such as ectoin and
hydroxyectoin provide protection against drying, which can
be explored for cosmetic applications.[96] Methanotrophic
bacteria can remove a significant amount of methane, which
has been applied in the production of materials and
bioremediation in recent years.[97,98] The gaseous substrate
utilizers can utilize syngas to synthesize ethanol, acetate, 2,3‐
butanediol, and PHA in bioprocessing.[99,100] As gas is the
only C‐source for those gaseous utilizers, there is a minor
possibility of contamination during the process.[99] The
ability to consume cellulose by Paenibacillus tarimensis allows
it to be an optimal candidate for low‐cost cellulose substrates,
demonstrating its potential for saccharification of lignocellu-
lose in biorefinery processes.[101] Adaptation to high con-
centrations of heavy metals such as Cu2+, Zn2+ allows
metalophiles to thrive in metal‐polluted sites, providing
opportunities for bioremediation and biomining under open
conditions.[102] Symbiotic microorganisms prone to form
mixed‐species biofilms are useful for immobilizing cells
under continuous fermentations.[103,109]
Compared with commonly used mesophilic microorgan-
isms, the challenges of utilizing extremophiles could be
summarized as follows: i) requirements to study and test the
optimum conditions for culturing these microorganisms; ii) the
development of bioreactors composed of materials that with-
stand the extreme conditions required for extremophiles
cultivation; iii) the genome information of newly isolated
species are unknown which limits the process of metabolic
engineering; and iv) their enzymes or useful products need be
characterized for further research and application. It is

Table 2. Potential contamination‐resistant microorganisms as platform strains for the NGIB.

Strains Growth properties References

Acidophiles or alkalophiles Low or high pH (pH < 5 or >8.5) [65,93]

Psychrophiles or thermophiles Low or high temperature (T < 25 °C or T > 45 °C) [94,95]

[96]
Xerophiles Able to grow on reduced water activities (such as high sugar concentration)
[97,98]
Methanotrophs Utilization of methane as a sole C‐source
[99,100]
Gaseous substrate utilizers Able to use H2, CO, CO2 as energy and/or C‐source
[101]
Cellulose or chitin utilizers Able to consume cellulose or chitin as a sole C‐source
Metalophiles Able to grow in the presence of high concentrations of heavy metal such as Cu2+, Zn2+, and so forth [102]

[103]
Symbiotic microorganisms Depend on each other for growth
[68,104]
Halophiles Able to grow on high NaCl concentration

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Figure 3. Engineering the halophilic Halomonas spp. for developing NGIB. A) Tools B) methods and C) the production of multiple products: a) Porin
promoter expression system; b) T7‐like expression system; c) CRISPRi engineering system; d) CRISPR/Cas9 engineering system; e) Increasing oxygen
availability; f) Morphology engineering; g) High‐cell‐density growth; h) Large‐scale fermentation; i) PHA product types: PHB, PHBV, and P3HB4HB; j)
Other products: PhaP, PhaR, ALA, and ectoine. gRNA, guide RNA; PHBV, poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate); RBS, ribosome binding site;
sgRNA, single‐guide RNA; VHb, Vitreoscilla hemoglobin.

necessary to overcome these drawbacks for developing extre-
mophiles as ideal platforms for NGIB.
6.5. Halomonas spp. as a Platform for NGIB
Endowed with the unique ability of rapid growth at high salt
concentrations and alkaline pH, two halophiles, namely Halomonas
bluephagenesis (H. bluephagenesis) and H. campaniensis, have been
successfully developed as the platform strains for NGIB due to their
ability to rapidly grow in a contamination‐free under open‐ and
continuous‐fermentation process.[64,68,110] H. bluephagenesis, isolated
from Aydingol Lake of Xinjiang/China in the authors’ laboratory,
has been studied with an available whole‐genome sequence and
engineering tools to be reconstructed for the productions of various
valuable chemicals and materials.[66,72,111,112]
Several molecular engineering tools for Halomonas spp. have
been developed (Figure 3A,B).[113] A constitutive chromosomal
expression system based on highly expressed porin protein gene
porin[114] and an inducible cassette named T7‐like expression system
induced by isopropyl‐β‐D‐thiogalactoside (IPTG) via expressing
RNA polymerases (RNAP) under a lacI‐controlled Ptac promoter
have been exploited.[115] As powerful tools for gene engineering in
many model organisms, clustered regularly interspaced short
palindromic repeats interference (CRISPRi)[116] and CRISPR/Cas9
system[112] have been successfully exploited and adopted in
Halomonas spp. Other method such as morphology engineering,[72]
effective expression system for increasing oxygen availability,[71]
controlling of NADH/NAD+ ratio,[117] and promoter engineer-
ing[118] for enhancing the accumulation of PHA in Halomonas spp.
have also been developed.
Halomonas spp. has been exploited as a suitable NGIB
platform for the production of a variety of chemicals including
PHA, proteins, ectoine, and other high value‐added chemicals
(Figure 3C).[63,68] Wild‐type H. bluephagenesis reached 80 g L−1
cell dry weight (CDW) containing approximately 80% PHB
during a 56 h fed‐batch process.[66] The engineered Halomonas
TD01 achieved 80 g L−1 CDW containing 70 wt% P(3HB‐co‐
8 mol% 3HV).[110] Recently, a strain named H. bluephagenesis
TDHCD‐R3‐8‐3 with an ablility to grow to a high‐cell‐density
was constructed and demonstrated to reach more than 90 g L−1
CDW containing 79% PHA under continuous fermentation
conditions;[119] H. bluephagenesis TD40 even reached 100 g L−1
CDW containing 60% poly(3‐hydroxybutyrate‐co‐4‐hydroxybu-
tyrate) (P3HB4HB) in a 5 m3 fermenter.[120] A variety of
enzymes useful in the food industry like amylases and
cellulases were produced by Halomonas spp.[121,122] Two PHA
synthesis regulatory proteins, PhaR and PhaP, which have
amphiphilic property were produced as biosurfactants with
good emulsification ability by engineered Halomonas
spp.[110,123] Ectoin can also be produced by halophile bacterium
Chromohalobacter salexigens at a high‐cell‐density weighting up
to 61 g L−1, 540 mg ectoine g−1 cells from two continuously
operated bioreactors.[124] Other high value‐added chemicals like
5‐aminolevulinic acid (ALA) and coproduction of ALA and PHB
were realized in engineered Halomonas spp.[125]
7. Conclusions
Development of sustainable technologies is urgent for realizing
the shift from chemical industry to bio‐based production.
Industrial biotechnology has already established available
routes to both natural and synthetic products as an alternative
technology and its role will continue to grow. Since sterilization
is necessary for CIB, it should be avoided in NGIB which

Biotechnol. J. 2019, 14, 1800437 1800437 (7 of 9) © 2019 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
reduces processes complexity, water and energy consumption
as well as investment cost. The unique scientific and
technological properties of NGIB will offer more opportunities
for making bioprocess more cost‐effective and operation
friendly. As one of the potential chassis for NGIB, Halomonas
spp. has been successfully engineered to produce a variety of
products under open nonsterile and continuous conditions. It is
expected that such extremophilic bacteria will become robust
chassis for NGIB to produce more competitive products.
Further breakthroughs in halophiles and other extremophiles
will lead to their commercialization. The future of NGIB based
on these microorganisms looks very promising in transforming
the CIB into more competitive processes.
Acknowledgements
This research was financially supported by grants from National Natural
Science Foundation of China (Grant No. 31870859, 21761132013, and
31430003), the Tsinghua President Fund (Grant No. 2015THZ10) and
National Postdoctoral Program for Innovative Talents (Grant No.
BX201700130).
Conflict of Interest
The authors declare no conflict of interest.
Keywords
extremophilic microorganisms, Halomonas, industrial biotechnology,
next‐generation industrial biotechnology, open continuous fermentation,
polyhydroxyalkanoates
Received: December 8, 2018
Revised: February 1, 2019
Published online: May 28, 2019
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